Production of 2-O-α-d-glucopyranosyl-l-ascorbic acid using sucrose phosphorylase by semi-rational design

2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) is often substituted for l-ascorbic acid (L-AA) in health- and skincare products due to its enhanced stability and comparable antioxidant. Enzymatic catalysis for AA-2G is gaining widespread interest and sucrose phosphorylases (SPase) have shown promise in achieving higher yields. To enhance AA-2G synthesis, we screened and identified the SPase from Bifidobacterium longum (BlSPase) as the starting enzyme, with an optimal pH of 5.4 and temperature of 45 °C for AA-2G synthesis. Subsequently, we conducted semi-rational design on BlSPase based on modeling and molecular docking. L-AA was docked with the BlSPase model to analyze key residues in the 'loop-door' domain and substrate-binding pocket. Through alanine scanning and saturation mutagenesis, a mutant library was created. After single-point and composite mutation, L341V/V346P was identified as the optimal variant. Ultimately, within 72 h, we achieved yield of 358.6 g/L AA-2G with a molar conversion of L-AA reaching 75.7 %.